Pancreatic islet transplantation provides a promising therapy for Type 1 Diabetes mellitus (Tl DM), in which the majority of pancreatic insulin-producing p-cells are destroyed. The shortage of transplantable donor islets has stimulated much interest in using human pluripotent stem cells such as human embryonic stem cells and human induced pluripotent stem cells (hiPSCs) as alternative, renewable sources to generate functional B-cells. Our long-term goal is to develop an autologous cell-based therapy to replenish insulin producing B-cells for the purpose of treating T1 DM by inducing the differentiation of hiPSCs. The remarkable developmental and differentiation potential of hiPSCs makes them attractive candidates for cell-based therapies. Before the full potential of hiPSCs can be realized, however, it is necessary to understand the complex signaling and genetic mechanisms that control their differentiation. This proposal will employ hiPSC as an exciting new model to dissect the molecular and cellular programs that regulate human B-cells differentiation. Through use of a novel TALEN technology to make knock-in reporter hiPSC lines, the proposed research will develop methods for isolating human B-cells progenitors and tracing the fates of their differentiated progeny. The proposed studies are aimed at defining and characterizing late stage progenitors of B-cell development, establishing their lineage relationships, and identifying cell type-specific cell surface markers of these progenitor populations and signaling pathways that guide their fate. These will be addressed by pursuing three Specific Aims: 1) Develop cell type-specific reporter hiPSCs for ordering events in human B-cells differentiation. 2) Determine the roles of Wnt, Notch and other critical signaling pathways in human B-cells differentiation. 3) Establish the lineage relationships and differentiation potentials of hiPSC derived B-cell sprogenitor subsets. Using genetic, cell biological and in vivo approaches, the planned research will improve our understanding of the molecular and cellular programs underlying human B-cells differentiation, maturation and function.